The present invention is in support of the development of an oleaginous yeast that accumulates oils enriched in long-chain ω-3 and/or ω-6 polyunsaturated fatty acids (“PUFAs”; e.g., 18:3, 18:4, 20:3, 20:4, 20:5, 22:6 fatty acids). Toward this end, the natural abilities of oleaginous yeast (mostly limited to 18:2 fatty acid production) have been enhanced by advances in genetic engineering, leading to the production of 20:4 (arachidonic acid or “ARA”), 20:5 (eicosapentaenoic acid or “EPA”) and 22:6 (docosahexaenoic acid or “DHA”) PUFAs in transformant Yarrowia lipolytica. These ω-3 and ω-6 fatty acids were produced by introducing and expressing heterologous genes encoding the ω-3/ω-6 biosynthetic pathway in the oleaginous host (see co-pending U.S. patent application Ser. No. 10/840,579 and No. 60/624,812, each entirely incorporated herein by reference). However, in addition to developing techniques to introduce the appropriate fatty acid desaturases and elongases into these particular host organisms, it is also necessary to increase the transfer of PUFAs into storage lipid pools following their synthesis.
As is well known in the art, the process of triacylglycerol (TAG) biosynthesis (wherein newly synthesized PUFAs are transferred into a host organism's storage lipid pools) requires the catalytic activity of various acyltransferases as most free fatty acids become esterified to coenzyme A (CoA) to yield acyl-CoAs. Specifically, a series of four reactions occur in the endoplasmic reticulum of the cell to form TAGs, as shown in the Table below.
TABLE 1General Reactions Of de Novo Triacylglycerol BiosynthesisReactionEnzymesn-Glycerol-3-Glycerol-3-phosphate acyltransferase (GPAT);Phosphate →[E.C. 2.3.1.15]; esterifies 1st acyl-CoA to sn-1Lysophosphatidicposition of sn-glycerol 3-phosphateAcid (1-acyl-sn-glycerol 3-phosphate or“LPA”)LPA →Lysophosphatidic acid acyltransferase (LPAAT)Phosphatidic Acid[E.C. 2.3.1.51]; esterifies 2nd acyl-CoA to sn-2(1,2-diacylglycerolposition of LPAphosphate or “PA”)PA → 1,2-Phosphatidic acid phosphatase [E.C. 3.1.3.4]Diacylglycerolremoves a phosphate from PA(“DAG”)DAG →Diacylglycerol acyltransferase (DGAT) [E.C.Triacylglycerol2.3.1.20]; transfers acyl-CoA to the sn-3 position of(“TAG”)DAGOrPhospholipid:diacylglycerol acyltransferase (PDAT)[E.C.2.3.1.158]; transfers fatty acyl-group from sn-2positionof phosphatidylcholine to sn-3 position ofDAGIn addition to those acyltransferases above, acyl-CoA:cholesterol acyltransferases (ACATs), lecithin:cholesterol acyltransferases (LCATs) and lysophosphatidylcholine acyltransferases (LPCATs) are also intimately involved in the biosynthesis of TAGs. The role of each of these acyltransferases in regulating lipid acyl composition is largely mediated through their individual substrate specificities.
This application is concerned primarily with the second step in the synthesis of TAG (wherein LPA is converted to PA) limits the acyltransferase(s) of primary importance to LPAAT (also referred to as acyl-CoA:1-acyl-sn-glycerol-3-phosphate 2-O-acyltransferase, 1-acyl-sn-glycerol-3-phosphate acyltransferase, AGAT and/or 1-acylglycerolphosphate acyltransferase in the literature). By inspection of the LPAAT activities in isolated membranes from seed tissues, it has been shown that LPAAT specificities vary from species to species in accordance with the kinds of fatty acyl groups found in the sn-2 positions of the respective storage oils. Thus, the acyl-CoA specificity of LPAAT can dramatically affect the types of fatty acyl groups found in the sn-2 position of plant oils. Similarly, WO 2004/087902 (Example 6) compared the activity of LPAAT in microsomal membranes of the filamentous fungus Mortierella alpina to that of flax and sunflower. These results suggest that the M. alpina LPAAT displays a wide specificity for acyl-CoAs, which is in contrast to the LPAATs of flax and sunflower. Subsequently, two Mortierella alpina LPAATs (GenBank Accession Nos. CQ891250 and CQ891252, were isolated and expressed in Saccharomyces cerevislae. 
Although similar empirical data concerning the Yarrowia lipolytica LPAAT substrate specificity and its effect on final TAG composition is lacking, wildtype Y. lipolytica's inability to produce anything other than a 18:2 fatty acid suggests a need for a heterologous LPAAT gene.
Despite the identification and public disclosure of several genes coding for LPAAT from various bacteria, yeast and plants, few genes are available from those microorganisms that naturally produce long-chain PUFAs (e.g., Mortierella, Pythium, Cyclotella, Nitzschia, Crypthecodinium and Thraustochytrium, producing e.g., ARA, EPA and/or DHA). Although it is likely that many of these organisms possess genes encoding LPAATs that would be preferred for the incorporation of long-chain PUFAs (i.e., relative to a LPAAT that does not naturally interact with long-chain PUFAs), the only known disclosure providing genes encoding LPAATs from these types of organisms is that of WO 2004/087902. Thus, there is a need for the identification and isolation of a gene encoding LPAAT from an organism such as those suggested above, to permit its use in the production and accumulation of long-chain PUFAs in the storage lipid pools (i.e., TAG fraction) of transformant oleaginous yeast.
Surprisingly, the Applicants have isolated a novel gene from the filamentous fungus Mortierella alpina that is a LPAAT homolog. This gene is clearly differentiated from those M. alpina LPAAT sequences provided in the art. It is expected that the gene of the present invention (“LPAAT2”) will be useful to enable one to modify the transfer of long-chain free fatty acids (e.g., ω-3 and/or ω-6 fatty acids) into the TAG pool in oleaginous yeast.